WO2004035139A1 - Regulation de mkk3 - Google Patents

Regulation de mkk3 Download PDF

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Publication number
WO2004035139A1
WO2004035139A1 PCT/GB2003/004477 GB0304477W WO2004035139A1 WO 2004035139 A1 WO2004035139 A1 WO 2004035139A1 GB 0304477 W GB0304477 W GB 0304477W WO 2004035139 A1 WO2004035139 A1 WO 2004035139A1
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WIPO (PCT)
Prior art keywords
polypeptide
mkk3
expression
activity
agent
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PCT/GB2003/004477
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English (en)
Inventor
Kevin Bruce Bacon
Herath Mudiyanselage Athula Chandrasiri Herath
Ningshu Liu
Christian Rohlff
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Oxford Glycosciences (Uk) Limited
Bayer Healthcare Ag
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Application filed by Oxford Glycosciences (Uk) Limited, Bayer Healthcare Ag filed Critical Oxford Glycosciences (Uk) Limited
Priority to AU2003274328A priority Critical patent/AU2003274328A1/en
Publication of WO2004035139A1 publication Critical patent/WO2004035139A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/48—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase
    • C12Q1/485—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase involving kinase
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/005—Enzyme inhibitors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A61P11/06—Antiasthmatics
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/04—Screening involving studying the effect of compounds C directly on molecule A (e.g. C are potential ligands for a receptor A, or potential substrates for an enzyme A)

Definitions

  • the present invention relates to methods for the treatment of inflammatory diseases including respiratory diseases, such as asthma and asthma related conditions, comprising regulation of the polypeptide mitogen-activated protein kinase kinase 3 (MKK3), agents which modulate the expression or activity of the polypeptide, methods for the identification of such agents and the use of MKK3 in the diagnosis of inflammatory diseases including respiratory diseases such as asthma and asthma related conditions.
  • MKK3 mitogen-activated protein kinase kinase 3
  • Asthma is an inflammatory disease characterised by obstructed airflow. It is estimated to affect 5-10% of the world's population, and to be increasing in prevalence across the majority of age groups.
  • Asthma is thought to arise as a result of interactions between multiple genetic and environmental factors and is characterised by three major features: 1) intermittent and reversible airway obstruction caused by bronchoconstriction, increased mucus production, and thickening of the walls of the airways that leads to a narrowing of the airways, 2) airway hyper-responsiveness caused by a decreased control of airway calibre, and 3) airway inflammation.
  • Certain cells are critical to the inflammatory reaction of asthma; these include T cells and antigen presenting cells, B cells that produce IgE, and mast cells, basophils, eosinophils, and other cells that bind IgE.
  • effector cells accumulate at the site of allergic reaction in the airways and release toxic products that contribute to the acute pathology and eventually to the tissue destruction related to the disorder.
  • Other resident cells such as smooth muscle cells, lung epithelial cells, mucus-producing cells, and nerve cells may also be abnormal in individuals with asthma and may contribute to the pathology. While the airway obstruction of asthma, presenting clinically as an intermittent wheeze and shortness of breath, is generally the most pressing symptom of the disease requiring immediate treatment, the inflammation and tissue destruction associated with the disease can lead to irreversible changes that eventually make asthma a chronic disabling disorder requiring long term management.
  • the inflammation associated with asthma can lead to remodelling of the extracellular matrix, including sub-epithelial fibrosis, and it is this remodelling that is a major component of chronic asthma.
  • the extracellular matrix of the lung is a dynamic structure and tissue remodelling requires the controlled degradation of extracellular matrix proteins.
  • Net protease activity depends on the balance between proteases and protease inhibitors.
  • An imbalance between proteases and protease inhibitors such as elastase and alpha 1-antitrypsin (AAT) have been reported in the induced sputum from asthmatic patients relative to healthy control patients (Nignola A.M. et ah, 1998, Am. J. Respir. Crit. Med., 157, 2, 505-511).
  • Glycophorin A Cho & Sharon, 1992, Cell Immunol., 145:223-239
  • cyclosporin Alexander et ah, 1992, Lancet, 339:324-328
  • a nonapeptide fragment of IL-2 Zav'yalov et al., 1992, Immunol. Lett., 31:285-288
  • cyclosporin is used as an immunosuppressant after organ transplantation. While these agents may represent alternatives to steroids in the treatment of asthmatics, they inhibit interleukin-2 dependent T lymphocyte proliferation and potentially critical immune functions associated with homeostasis.
  • mast cell stabilisers such as cromolyn or leukotriene receptor inhibitors such as montelukast
  • montelukast leukotriene receptor inhibitors
  • Therapies in trials at the moment include recombinant anti-proteinases (alpha 1-antitrypsin, AAT) or small molecules to proteases (AAT); antibodies or biological receptors to cytokines or key inflammatory cells; immune-based therapy; and inflammatory antagonists (VLA-4 antagonists, selectin antagonists).
  • AAT recombinant anti-proteinases
  • AAT small molecules to proteases
  • VLA-4 antagonists inflammatory antagonists
  • COPD chronic obstructive pulmonary
  • COPD chronic obstructive pulmonary
  • This is a condition defined physiologically as airflow obstruction that generally results from a mixture of emphysema and peripheral airway obstruction due to chronic bronchitis (Senior R.M. & Shapiro S.D., Chronic obstructive pulmonary disease: Epidemiology, pathophysiology, and pathogenesis. In Pulmonary Diseases and Disorders, 3rd ed., New York, McGraw-Hill, 1998, pp. 659- 681; Barnes P.J., Mechanisms in COPD.
  • Mitogen-activated protein kinase kinase 3 (MKK3) is activated by phosphorylation on ser-189 and thr-193 and catalyses the phosphorylation of a threonine and a tyrosine residue in the mitogen- activated protein (MAP) kinase p38, resulting in its activation.
  • MAP mitogen- activated protein
  • Expression of M K3 in cultured cells causes a selective increase in p38 MAP kinase activity.
  • Pro-inflammatory cytokines and environmental stress trigger this activation of the p38 MAP kinase pathway.
  • the sequence of MKK3 can be found under the accession number P46734 in the SwissProt database (available at www.expasy.org) and is shown in Figure 1.
  • MKK3 has three splice variants A (SEQ ID NO: 1), B (SEQ ID N0:4) and C (SEQ ID N0:3).
  • the current invention is based on the finding that MKK3 represents an approach to therapeutic intervention in inflammatory diseases including respiratory diseases such as asthma and asthma related conditions.
  • MKK3 is differentially expressed in lymphocytes from asthma patients compared to normal volunteers. In particular, an increase in MKK3 expression was seen in CD8 lymphocytes from asthma patients compared to normal subjects.
  • MKK3 may be a suitable target for inflammatory diseases, in particular asthma therapy and diagnosis.
  • one aspect of the invention provides a method of prophylaxis and/or treatment of an inflammatory disease which comprises administering to a subject in need thereof a therapeutically effective amount of an agent which modulates the expression or activity of a MKK3 polypeptide.
  • the invention also provides for the use of an agent which modulates the expression and/or activity of a MKK3 polypeptide for the prophylaxis and/or treatment of an inflammatory disease.
  • the invention also provides the use of an agent which modulates the expression or activity of a MKK3 polypeptide in the manufacture of a medicament for the treatment of an inflammatory disease.
  • MKK3 polypeptide is variant C.
  • MK 3 variant C can be used as a target for the treatment of inflammatory diseases such as respiratory diseases, e.g. asthma, and modulators of MKK3 variant C can be used for the treatment of such diseases.
  • the inflammatory disease to be treated according to the method of the invention is preferably a respiratory disease, especially asthma.
  • inhibitors of MKK3 may have function in inhibiting the development of asthma pathology.
  • agents for use in the methods of the invention preferably include, without limitation: i) an agent that modulates MKK3 activity, e.g. an inhibitor of MKK3; ii) an agent that regulates molecules upstream of MKK3; or iii) an agent that regulates, preferably decreases the expression of MKK3, such as siRNA.
  • MKK3 polypeptides include polypeptides which:
  • variants having one or more amino acid substitutions, modifications, deletions or insertions relative to the amino acid sequence selected of SEQ ID NO: 1, 3 or 4, provided that such variants exhibit the activity of a MKK3 polypeptide.
  • polypeptides described in (i) and (ii) above are hereinafter referred to as "MKK3 polypeptides".
  • polypeptides includes peptides, polypeptides and proteins. These are used interchangeably unless otherwise specified.
  • a further aspect of the invention provides methods of screening for anti-inflammatory agents that modulate the expression or activity of a MKK3 polypeptide.
  • a method of screening for anti-inflammatory agents that modulate the expression and/or activity of a MKK3 polypeptide comprising: a) contacting a MKK3 polypeptide or a system capable of expressing said polypeptide with a candidate agent; and b) determining whether the candidate agent causes the expression and/or activity of said polypeptide or the expression of a nucleic acid encoding said polypeptide, to change.
  • the expression and/or activity of a MKK3 polypeptide or the expression of a nucleic acid molecule encoding said polypeptide is compared with a predetermined reference range.
  • the method further comprises selecting an agent, which modulates the expression and/or activity of a MKK3 polypeptide or the expression of a nucleic acid molecule encoding said polypeptide, for further testing for use in the prophylaxis and/or treatment of an inflammatory disease such as asthma.
  • the invention thus provides assays for use in drug discovery in order to identify or verify the efficacy of agents for the prophylaxis and/or treatment of an inflammatory disease.
  • Active agents identified using these methods can be used as lead agents for drug discovery, or used therapeutically.
  • Expression of a MKK3 polypeptide can be assayed by, for example, immunoassays, gel electrophoresis followed by visualisation, detection of mRNA or MKK3 polypeptide activity, (Peters G. et al, 2003, Biochimie, 85, 527-534) or any other method taught herein or known to those skilled in the art.
  • Such assays can be used to screen candidate agents, in clinical monitoring or in drug development.
  • Candidate agents which may be tested for activity in the described assays include, but are not limited to, nucleic acids (e.g. DNA and RNA), carbohydrates, lipids, polypeptides, peptides, peptidomimetics, agonists, small molecules and other drugs. Agents can be obtained using any of the numerous suitable approaches in combinatorial library methods known in the art, including: biological libraries; spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; the "one-bead one-compound” library method; and synthetic library methods using affinity chromatography selection.
  • the biological library approach is limited to peptide libraries, while the other four approaches are applicable to peptide, non-peptide oligomer or small molecule libraries of compounds (Lam, 1997, Anticancer Drug Des., 12:145; U.S. 5,738,996; and U.S. 5,807,683).
  • Active agents that interact with (e.g. bind to) a MKK3 polypeptide or nucleic acid molecule encoding said polypeptide may be identified in a cell-based assay system.
  • cells expressing a MKK3 polypeptide (or nucleic acid molecule encoding said polypeptide) are contacted with a candidate agent or a control agent and the ability of the candidate agent to interact with the MKK3 polypeptide (or nucleic acid molecule encoding said polypeptide) is determined.
  • this assay may be used to screen a plurality (e.g. a library) of candidate agents.
  • the cell for example, can be of prokaryotic origin (e.g. E.
  • the cells can express a MKK3 polypeptide (or nucleic acid molecule encoding said polypeptide) endogenously or be genetically engineered to express a MKK3 polypeptide (or nucleic acid molecule encoding said polypeptide).
  • the MKK3 polypeptide (or nucleic acid molecule encoding said polypeptide) or the candidate agent may be labelled, for example with a radioactive label (such as P, S or I) or a fluorescent label (such as fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde or fluorescamine) to enable detection of an interaction between a MKK3 polypeptide and a candidate agent.
  • a radioactive label such as P, S or I
  • a fluorescent label such as fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde or fluorescamine
  • the ability of the candidate agent to interact directly or indirectly with a MKK3 polypeptide (or nucleic acid molecule encoding said polypeptide) can be determined by methods known to those of skill in
  • the interaction between a candidate agent and a MKK3 polypeptide can be determined by flow cytometry, a scintillation assay, immunoprecipitation or western blot analysis.
  • Active agents that interact with (e.g. bind to) a MKK3 polypeptide or nucleic acid molecule encoding said polypeptide may be identified in a cell-free assay system.
  • a native or recombinant MKK3 polypeptide (or nucleic acid molecule encoding said polypeptide) or fragment thereof is contacted with a candidate agent or a control agent and the ability of the candidate agent to interact with a MKK3 polypeptide (or nucleic acid molecule encoding said polypeptide) is determined.
  • this assay may be used to screen a plurality (e.g. a library) of candidate agents.
  • a MKK3 polypeptide or nucleic acid molecule encoding said polypeptide
  • Said polypeptide or nucleic acid molecule may be partially or completely purified (e.g.
  • a MKK3 polypeptide may be a fusion protein comprising a MKK3 polypeptide or a biologically active portion thereof and a domain such as glutathionine-S-transferase.
  • a MKK3 polypeptide can be biotinylated using techniques well known to those of skill in the art (e.g. biotinylation kit, Pierce Chemicals; Rockford, IL). The ability of the candidate agent to interact with a MKK3 polypeptide can be can be determined by methods known to those of skill in the art.
  • a cell-based assay system may be used to identify active agents that bind to or modulate the activity of a MKK3 polypeptide, or a polypeptide such as an enzyme, or a biologically active portion thereof, which is responsible for the production of a MKK3 polypeptide or is responsible for the post- translational modification of said polypeptide.
  • a plurality e.g. a library
  • agents are contacted with cells that naturally or recombinantly express: (i) a MKK3 polypeptide; and (ii) a polypeptide that is responsible for processing of a MKK3 polypeptide, in order to identify agents that modulate the production, degradation, or post-translational modification of the polypeptide.
  • active agents identified in the primary screen can then be assayed in a secondary screen against cells naturally or recombinantly expressing the specific polypeptide of interest.
  • the ability of the candidate agent to modulate the production, degradation or post-translational modification of a polypeptide can be determined by methods known to those of skill in the art, including without limitation, flow cytometry, a scintillation assay, immunoprecipitation and Western blot analysis.
  • Active agents that competitively interact with (e.g. bind to) a MKK3 polypeptide may be identified in a competitive binding assay.
  • cells expressing the MKK3 polypeptide are contacted with a candidate agent and an agent known to interact with the MKK3 polypeptide; the ability of the candidate agent to competitively interact with the MKK3 polypeptide is then determined.
  • active agents that competitively interact with (e.g. bind to) a polypeptide may be identified in a cell-free assay system by contacting the polypeptide with a candidate agent and an agent known to interact with the polypeptide.
  • the ability of the candidate agent to interact with a polypeptide for use in the invention can be determined by methods known to those of skill in the art. These assays, whether cell-based or cell-free, can be used to screen a plurality (e.g. a library) of candidate agents.
  • Active agents that down-regulate the expression of a MKK3 polypeptide or nucleic acid molecule encoding said polypeptide may be identified by contacting cells (e.g. cells of prokaryotic origin or eukaryotic origin) expressing said polypeptide or nucleic acid molecule with a candidate agent or a control agent (e.g. phosphate buffered saline (PBS)) and determining the expression of a MKK3 polypeptide or nucleic acid molecule (e.g. mRNA encoding a MKK3 polypeptide).
  • a candidate agent or a control agent e.g. phosphate buffered saline (PBS)
  • the level of expression of the polypeptide or nucleic acid molecule in the presence of the candidate agent may be compared to the level of expression of the polypeptide or nucleic acid molecule encoding the polypeptide in the absence of the candidate agent (e.g. in the presence of a control agent).
  • the candidate agent can then be identified as a modulator of the expression of the polypeptide or nucleic acid molecule based on this comparison. For example, when expression of the polypeptide or nucleic acid molecule encoding the polypeptide is significantly lower in the presence of the candidate agent than in its absence, the candidate agent is identified as an inhibitor of expression of the polypeptide nucleic acid molecule.
  • the level of expression of a MKK3 polypeptide or nucleic acid molecule encoding said polypeptide can be determined by methods known to those of skill in the art based on the present description. For example, mRNA expression can be assessed by Northern blot analysis or RT-PCR, and polypeptide levels can be assessed by Western blot analysis.
  • Active agents that modulate the activity of a MKK3 polypeptide may be identified by contacting a preparation containing said polypeptide, or cells (e.g. prokaryotic or eukaryotic cells) expressing said polypeptide with a candidate agent or a control agent and determining the ability of the candidate agent to modulate (e.g. up-regulate) the activity of a MKK3 polypeptide.
  • the activity of a MKK3 polypeptide can be assessed by detecting its effect on a "downstream effector" for example, but without limitation, detecting catalytic or enzymatic activity of the target on a suitable substrate (e.g.
  • phosphor-ZAP or p-nitrophenyl phosphate see Yong-Jiu Jin et al, 1999, J. Biol. Chem., 274; Cragges G. & Kellie S., 2001, J. Biol. Chem., 276; and Peters G.H. et al, 2003, Biochimie, 85, 527-534) or other techniques known to those of skill in the art can be used (see e.g. U.S. 5,401,639).
  • the candidate agent can then be identified as a modulator of the activity of a MKK3 polypeptide for use in the invention by comparing the effects of the candidate agent to the control agent.
  • Suitable control agents include phosphate buffered saline (PBS) and normal saline (NS).
  • agents that modulate the ability of a MKK3 polypeptide to activate p38 are identified by comparing p38 activity in the presence of the candidate agent and in the absence of the candidate agent (e.g. in the presence of a control agent). Such assays are well known in the art.
  • agents such as an enzyme, or a biologically active portion thereof, which is responsible for the production or degradation of a MKK3 polypeptide, or is responsible for the post-translational modification of a MKK3 polypeptide can be identified.
  • substantially pure native or recombinantly expressed MKK3 polypeptides or cellular extract containing native or recombinantly expressed MKK3 polypeptides are contacted with a plurality of candidate agents, for example but without limitation a plurality of agents presented as a library, that may be responsible for the processing of a MK 3 polypeptide, in order to identify such agents.
  • Active agents that modulate the expression, activity or both the expression and activity of a MKK3 polypeptide may be identified in an animal model.
  • suitable animals include, but are not limited to, mice, rats, rabbits, monkeys, guinea pigs, dogs and cats.
  • the animal used represents a model of the inflammatory disease of interest e.g. asthma.
  • the candidate agent or a control agent is administered (e.g. orally, by inhalation, rectally or parenterally such as intraperitoneally or intravenously) to a suitable animal and the effect on the expression, activity or both expression and activity of a MKK3 polypeptide is determined. Changes in the expression of a polypeptide can be assessed by any suitable method described above.
  • MKK3 polypeptides may also be used as a "bait protein" in a two-hybrid assay or three hybrid assay to identify other polypeptides that bind to or interact with said polypeptide (see, e.g. U.S. 5,283,317; Zervos et al.., 1993, Cell, 72:223-232; Madura et al, 1993, J. Biol. Chem., 268: 12046-12054; Bartel et al, 1993, Bio/Techniques, 14:920-924; Iwabuchi et al, 1993, Oncogene, 8:1693-1696; and WO 94/10300).
  • binding polypeptides are also likely to be involved in the propagation of signals by a MKK3 polypeptide as, for example, upstream or downstream elements of a signalling pathway involving said polypeptide.
  • polypeptides that interact with a MKK3 polypeptide can be identified by isolating a protein complex comprising a MKK3 polypeptide and identifying the associated polypeptides using methods known in the art such as mass spectrometry (for examples see Blackstock W. & Weir, M. 1999, Trends in Biotechnology, 17: 121-127; Rigaut G. 1999, Nature Biotechnology, 17: 1030-1032; Husi H. 2000, Nature Neurosci., 3:661-669; Ho Y. et al, 2002, Nature, 415:180-183; Gavin A. et al, 2002, Nature, 415: 141-147).
  • mass spectrometry for examples see Blackstock W. & Weir, M. 1999, Trends in Biotechnology, 17: 121-127; Rigaut G. 1999, Nature Biotechnology, 17: 1030-1032; Husi H. 2000, Nature Neurosci., 3:661-669; Ho Y. et al, 2002, Nature
  • MKK3 polypeptides may also be used in a method for the structure-based design of an agent, in particular a small molecule which acts to modulate the activity of said polypeptide, said method comprising:
  • the invention further provides novel active agents identified by the above-described methods and uses thereof for treatments as described herein.
  • agents which modulate the expression or activity of a MKK3 polypeptide find use in the treatment or prophylaxis of inflammatory diseases including respiratory diseases such as asthma.
  • the agents will generally be administered in the form of a pharmaceutical composition.
  • a pharmaceutical composition comprising an agent which modulates the expression or activity of a MKK3 polypeptide and a pharmaceutically acceptable diluent, excipient and/or carrier.
  • composition will usually be supplied as part of a sterile, pharmaceutical composition that will normally include a pharmaceutically acceptable carrier.
  • This composition may be in any suitable form, depending upon the desired method of administering it to a patient.
  • unit dosage form will generally be provided in a sealed container and may be provided as part of a kit.
  • a kit would normally, although not necessarily, include instructions for use. It may include a plurality of said unit dosage forms.
  • compositions may be adapted for administration by any appropriate route, for example by the oral (including buccal, sublingual), inhalation (e.g. via a fine powder formulation), nasal, rectal, topical (including buccal, sublingual or transdermal), vaginal or parenteral (including subcutaneous, intramuscular, intravenous or intradermal) route.
  • oral including buccal, sublingual
  • inhalation e.g. via a fine powder formulation
  • nasal, rectal e.g. via a fine powder formulation
  • topical including buccal, sublingual or transdermal
  • vaginal or parenteral including subcutaneous, intramuscular, intravenous or intradermal route.
  • parenteral including subcutaneous, intramuscular, intravenous or intradermal route.
  • compositions adapted for oral administration may be presented as discrete units such as capsules or tablets; as powders or granules; as solutions, syrups or suspensions (in aqueous or non- aqueous liquids; or as edible foams or whips; or as emulsions).
  • compositions adapted for nasal administration wherein the carrier is a solid include a coarse powder having a particle size for example in the range 20 to 500 microns which is administered by rapid inhalation through the nasal passage from a container of the powder held close up to the nose.
  • Suitable compositions wherein the carrier is a liquid, for administration as a nasal spray or as nasal drops, include aqueous or oil solutions of the active agent.
  • Suitable excipients for tablets or hard gelatine capsules include lactose, maize starch or derivatives thereof, stearic acid or salts thereof.
  • Suitable excipients for use with soft gelatine capsules include for example vegetable oils, waxes, fats, semi-solid, or liquid polyols etc.
  • excipients which may be used include for example water, polyols and sugars.
  • oils e.g. vegetable oils
  • compositions adapted for transdermal administration may be presented as discrete patches intended to remain in intimate contact with the epidermis of the recipient for a prolonged period of time.
  • the active ingredient may be delivered from the patch by iontophoresis as generally described in Pharmaceutical Research, 3(6):318 (1986).
  • Compositions adapted for topical administration may be formulated as ointments, creams, suspensions, lotions, powders, solutions, pastes, gels, sprays, aerosols or oils.
  • compositions are preferably applied as a topical ointment or cream.
  • the active ingredient may be employed with either a paraffmic or a water-miscible ointment base.
  • the active ingredient may be formulated in a cream with an oil-in-water cream base or a water-in-oil base.
  • compositions adapted for topical administration to the eye include eye drops wherein the active ingredient is dissolved or suspended in a suitable carrier, especially an aqueous solvent.
  • compositions adapted for topical administration in the mouth include lozenges, pastilles and mouth washes.
  • Compositions adapted for rectal administration may be presented as suppositories or enemas.
  • compositions adapted for administration by inhalation include fine particle dusts or mists which may be generated by means of various types of metered dose pressurised aerosols, nebulisers or insufflators.
  • agents of the instant invention when they are to be used in the treatment of asthma, they will be formulated as aerosols.
  • aerosol includes any gas-borne suspended phase of the agent of the invention which is capable of being inhaled into the bronchioles or nasal passages.
  • aerosol includes a gas-borne suspension of droplets of the agents of the instant invention, as may be produced in a metered dose inhaler or nebuliser, or in a mist sprayer.
  • Aerosol also includes a dry powder composition of an agent of the instant invention suspended in air or other carrier gas, which may be delivered by insufflation from an inhaler device, for example.
  • the preferred range of concentration of the agents of the invention is 0.1-100 milligrams (mg)/ milliliter (ml), more preferably 0.1-30mg/ml, and most preferably, 1-10 mg/ml.
  • the solutions are buffered with a physiologically compatible buffer such as phosphate or bicarbonate.
  • the usual pH range is 5 to 9, preferably 6.5 to 7.8, and more preferably 7.0 to 7.6.
  • Suspensions of the agents of the present invention in hydrofluoroalkane propellants especially 1,1,1,2-tetrafluoroethane or 1,1,1,2,3,3,3- heptafluoropropane, optionally in the presence of a surfactant and/or cosolvent (e.g. ethanol) in a pressurised canister may also be provided with a suitable delivery device for the treatment of asthma.
  • a surfactant and/or cosolvent e.g. ethanol
  • such methods comprise pressurising or providing a means of pressurising a container of the solution, usually with an inert carrier gas, and passing the pressurised gas through a small orifice, thereby pulling droplets of the solution into the mouth and trachea of the patient to which the drug is to be administered.
  • a mouthpiece is fitted to the outlet of the orifice to facilitate delivery into the mouth and trachea.
  • Solutions of the active agents may be administered by any of the conventional means for creating aerosols in asthma medication, such as metered dose inhalers, jet nebulizers, or ultrasonic nebulizers.
  • such device may include a mouthpiece fitted around the orifice.
  • a solution of an active agent may be administered using a nasal sprayer.
  • the active agents may also be administered using a dry powder inhaler containing a dry powder comprising an active agent optionally with an excipient.
  • compositions adapted for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams or spray compositions.
  • Compositions adapted for parenteral administration include aqueous and non-aqueous sterile injection solution which may contain anti-oxidants, buffers, bacteriostats and solutes which render the composition substantially isotonic with the blood of the intended recipient; and aqueous and non- aqueous sterile suspensions which may include suspending agents and thickening agents.
  • Excipients that may be used for injectable solutions include water, alcohols, polyols, glycerine and vegetable oils, for example.
  • compositions may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilised) condition requiring only the addition of the sterile liquid carried, for example water for injections, immediately prior to use.
  • Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets.
  • the compositions may contain preserving agents, solubilising agents, stabilising agents, wetting agents, emulsifiers, sweeteners, colorants, odourants, salts, buffers, coating agents or antioxidants. They may also contain therapeutically active agents in addition to the agent which increases the expression and/or activity of a MKK3 polypeptide, e.g.
  • additional agents for the prophylaxis and/or treatment of inflammatory diseases e.g. asthma.
  • inflammatory diseases e.g. asthma.
  • the optimal quantity and spacing of individual dosages of the active agents will be determined by the nature and extent of the condition being treated, the form, route and site of administration, and the particular mammal being treated, and that such optimums can be determined by conventional techniques.
  • the optimal course of treatment i.e. the number of doses of the active agent given per day for a defined number of days, can be ascertained by those skilled in the art using conventional course of treatment determination tests.
  • compositions of the invention also can be administered in combination therapy, i.e. combined with other agents.
  • the combination therapy can include a composition of the present invention with at least one other anti-inflammatory agent or other conventional therapy.
  • MKK3 polypeptides or variants thereof for use in the screening methods of the invention may be provided in isolated or recombinant form, and may be fused to other moieties.
  • fusions of the polypeptides, derivatives or fragments thereof with localisation-reporter polypeptides such as the Green Fluorescent Protein (U.S. 5,625,048, 5,777,079, 6,054,321 and 5,804,387) or the DsRed fluorescent protein (Matz M.N., et al, Nature Biotech., 17:969-973) are specifically contemplated.
  • the polypeptides, or variants thereof may be provided in substantially pure form, that is to say free, to a substantial extent, from other polypeptides.
  • a MKK3 polypeptide may be provided in a composition in which it is the predominant component present, (i.e. it is present at a level of at least 50%; preferably at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%; when determined on a weight/weight basis excluding solvents or carriers).
  • a MKK3 polypeptide may comprise or consist of the particular amino acid sequence of SEQ ID NO: 1, 3 or 4 or may have an additional N-terminal and/or an additional C-terminal amino acid sequence relative to the sequence given in SEQ ID NO: 1, 3 or 4. Additional N-terminal or C-terminal sequences may be provided for various reasons. Techniques for providing such additional sequences are well known in the art.
  • Additional sequences may be provided in order to alter the characteristics of a polypeptide. This can be useful in improving expression or regulation of expression in particular expression systems.
  • an additional sequence may provide some protection against proteolytic cleavage. This has been done for the hormone somatostatin by fusing it at its N-terminus to part of the ⁇ galactosidase enzyme (Itakwa et ⁇ Z, 1977, Science, 198: 105-63).
  • a fusion protein may be provided in which a polypeptide is linked to a moiety capable of being isolated by affinity chromatography.
  • the moiety may be an antigen or an epitope and the affinity column may comprise immobilised antibodies or immobilised antibody fragments that bind to said antigen or epitope (desirably with a high degree of specificity).
  • the fusion protein can usually be eluted from the column by addition of an appropriate buffer.
  • N-terminal or C-terminal sequences may, however, be present simply as a result of a particular technique used to obtain a polypeptide and need not provide any particular advantageous characteristic to the polypeptide.
  • Such polypeptides are within the scope of the present invention.
  • the resultant polypeptide should exhibit the biological activity of a MKK3 polypeptide having the amino acid sequence shown in SEQ ID NO: 1, 3 or 4.
  • variants (derivatives) of MKK3 polypeptides it will be appreciated by the person skilled in the art that these polypeptides are variants of MKK3 polypeptides given in SEQ ID NO: 1, 3 or 4, provided that such variants exhibit the biological activity of a MKK3 polypeptide.
  • variant refers to a polypeptide that comprises an amino acid sequence of a parent polypeptide that has been altered by the introduction of amino acid residue substitutions, deletions or additions, and/or amino acid modifications such as but not limited to, phosphorylation and glycosylation.
  • variants can include post-translational modifications, for example but without limitation, phosphorylation, glycosylation and farnesylation.
  • Alterations in the amino acid sequence of a polypeptide can occur which do not affect the activity of the polypeptide. These include amino acid deletions, insertions and substitutions and can result from alternative splicing and/or the presence of multiple translation start sites and stop sites.
  • Polymorphisms may arise as a result of the infidelity of the translation process. Thus changes in amino acid sequence may be tolerated which do not affect the polypeptide' s biological or immunological activity.
  • amino acids have similar properties.
  • One or more such -imino acids of a polypeptide can often be substituted by one or more other such amino acids without eliminating a desired activity of that polypeptide.
  • amino acids glycine, alanine, valine, leucine and isoleucine can often be substituted for one another (amino acids having aliphatic side chains).
  • amino acids having aliphatic side chains amino acids having aliphatic side chains.
  • amino acids which can often be substituted for one another include: - phenylalanine, tyrosine and tryptophan (amino acids having aromatic side chains);
  • cysteine and methionine amino acids having sulphur-containing side chains
  • - asparagine and glutamine can substitute for phospho-serine and phospho-threonine, respectively
  • Amino acid deletions or insertions may also be made relative to the amino acid sequence given in SEQ ID NO: 1, 3 or 4.
  • amino acids that do not have a substantial effect on the biological and/or immunological activity of the polypeptide, or at least which do not eliminate such activity may be deleted.
  • Such deletions can be advantageous since the overall length and the molecular weight of a polypeptide can be reduced whilst still retaining activity. This can enable the amount of polypeptide required for a particular purpose to be reduced.
  • Amino acid insertions relative to the sequence given in SEQ ID NO: 1, 3 or 4 can also be made.
  • MKK3 polypeptide This may be done to alter the properties of a MKK3 polypeptide (e.g. to assist in identification, purification or expression, as explained in relation to fusion proteins).
  • Amino acid changes relative to the sequence given in SEQ ID NO: 1, 3 or 4 can be made using any suitable technique e.g. by using site-directed mutagenesis (Hutchinson et al, 1978, J. Biol. Chem., 253:6551).
  • amino acid substitutions or insertions to the MKK3 polypeptides can be made using naturally occurring or non-naturally occurring amino acids. Whether or not natural or synthetic amino acids are used, it is preferred that only L-amino acids are present.
  • preferred polypeptides have at least 50% sequence identity with a polypeptide as defined in SEQ ID NO:l, 3 or 4, more preferably the degree of sequence identity is at least 70%, at least 75%, at least 80%, at least 85%. Sequence identities of at least 90%, at least 95% or at least 98% are most preferred.
  • identity can be used to describe the similarity between two polypeptide sequences.
  • the degree of amino acid sequence identity can be calculated using a program such as "bestfit” (Smith & Waterman, Advances in Applied Mathematics, 482-489, 1981) to find the best segment of similarity between any two sequences.
  • the alignment is based on maximising the score achieved using a matrix of amino acid similarities, such as that described by Schwarz & Dayhof, 1979, Atlas of Protein Sequence and Structure, Dayhof M.O., Ed pp 353-358.
  • a software package well known in the art for carrying out this procedure is the CLUSTAL program. It compares the amino acid sequences of two polypeptides and finds the optimal alignment by inserting spaces in either sequence as appropriate.
  • Percentage identity is a well known concept in the art and can be calculated using, for example but without limitation, the BLASTTM software available fromNCBI (Altschul, S.F. et al, 1990, J. Mol. Biol. 215:403-410; Gish, W. & States, D.J. 1993, Nature Genet. 3:266-272. Madden, T.L. et al, 1996, Meth. Enzymol. 266:131-141; Altschul, S.F. et al, 1997, Nucleic Acids Res. 25:3389-3402); Zhang, J. & Madden, T.L. 1997, Genome Res. 7:649-656).
  • the BLASTX program aligns the largest stretch of similar sequence and assigns a value to the fit. For any one pattern comparison, several regions of similarity may be found, each having a different score.
  • two polypeptides of different lengths may be compared over the entire length of the longer fragment. Alternatively small regions may be compared. Normally sequences of the same length are compared for a useful comparison to be made.
  • a percent identity is defined as the number of identical amino acid residues as a percentage of the length of sequence of the polypeptide as defined in SEQ ID NO: 1, 3 or 4.
  • amino acid sequence where high degrees of sequence identity are present there will be relatively few differences in amino acid sequence. Thus for example they may be less than 20, less than 10, or even less than 5 differences.
  • Fragments of MKK3 polypeptides may be at least 10 amino acids in length, preferably they are at least 20, at least 30, at least 50 or at least 100 amino acids in length.
  • a fragment has at least 70% identity over its length to the amino acid sequence of SEQ ID NO: 1, 3 or 4, more preferably it has at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 98% identity.
  • polypeptides used in the present invention can be coded for by a large variety of nucleic acid molecules, taking into account the well-known degeneracy of the genetic code. They can be inserted into vectors and cloned to provide large amounts of DNA or RNA for further study. Suitable vectors may be introduced into host cells to enable the expression of polypeptides using techniques known to the person skilled in the art.
  • DNA constructs can readily be generated using methods well known in the art. These techniques are disclosed, for example in J. Sambrook et al, Molecular Cloning 2 nd Edition, Cold Spring Harbour Laboratory Press, 1989; in Old & Primrose, Principles of Gene Manipulation, 5th Edition, Blackwell Scientific Publications, 1994; and in Stryer, Biochemistry, 4th Edition, W.H. Freeman and Company, 1995. Modifications of DNA constructs and the polypeptides expressed such as the addition of promoters, enhancers, signal sequences, leader sequences, translation start and stop signals and DNA stability controlling regions, or the addition of fusion partners may then be facilitated.
  • the DNA construct will be inserted into a vector, which may be of phage or plasmid origin.
  • Expression of the polypeptide is achieved by the transformation or transfection of the vector into a host cell, which may be of eukaryotic or prokaryotic origin.
  • cell-free translation systems can be employed to produce recombinant polypeptides (e.g. rabbit reticulocyte lysate, wheat germ lysate, SP6/T7 in vitro T&T and RTS 100 E. Coli HY transcription and translation kits from Roche Diagnostics Ltd., Lewes, UK and the TNT Quick coupled Transcription/Translation System from Promega UK, Southampton, UK.
  • a polypeptide comprising an additional amino acid sequence which contains a signal sequlence, for example and without limitation, a signal peptide for secretion, or a leader sequence, a pro-sequence, a sequence to aid purification such as an affinity tag or an additional sequence conferring stability during production of the polypeptide.
  • sequences may be optionally removed as required.
  • a MKK3 polypeptide may be fused to other moieties including other polypeptides. Means for preparing such polypeptides are well known in the art.
  • Nucleotide sequences including DNA and RNA, and comprising a sequence encoding a MKK3 polypeptide, such as that shown in SEQ ID NO: 2, (or a variant thereof), may be synthesised using methods known in the art, such as using conventional chemical approaches or polymerase chain reaction (PCR) amplification. As a further alternative chemical synthesis may be used, this may be automated. Relatively short sequences may be chemically synthesised and ligated together to provide a longer sequence.
  • PCR polymerase chain reaction
  • MKK3 nucleic acids include nucleic acids which comprise or consist of: (i) comprises or consists of the DNA sequence shown in SEQ ID NO: 2 or its RNA equivalent; (ii) a sequence which codes for a derivative MKK3 polypeptide;
  • RNA equivalent when used above indicates that a given RNA molecule has a sequence which is complementary to that of a given DNA molecule, allowing for the fact that in RNA "U” replaces "T” in the genetic code.
  • the nucleic acid molecule may be in isolated, recombinant or chemically synthetic form.
  • MKK3 polypeptides may be expressed in glycosylated or non-glycosylated form. Non-glycosylated forms can be produced by expression in prokaryotic hosts, such as E. coll
  • Polypeptides comprising N-terminal methionine may be produced using certain expression systems, whilst in others the mature polypeptide will lack this residue.
  • Preferred techniques for cloning, expressing and purifying a MKK3 polypeptide are summarised below:
  • Polypeptides may be prepared natively or under denaturing conditions and then subsequently refolded.
  • Baculoviral expression vectors include secretory plasmids (such as pACGP67 from Pharmingen), which may have an epitope tag sequence cloned in frame (e.g. myc, N5 or His) to aid detection and allow for subsequent purification of the polypeptide.
  • Mammalian expression vectors may include pCD ⁇ A3 and pSecTag (both Invitrogen), and pREP9 and pCEP4 (Invitrogen).
  • E. coli systems include the pBad series (His tagged - Invitrogen) or pGex series (Pharmacia).
  • MKK3 nucleic acids can also be used to raise antibodies and for antisense therapy. Techniques for this are well-known by those skilled in the art, as discussed in more detail herein. In addition to nucleic acid molecules coding for MKK3 polypeptides referred to herein as
  • coding nucleic acid molecules also includes nucleic acid molecules complementary thereto.
  • both strands of a double stranded nucleic acid molecule may be included in the present invention (whether or not they are associated with one another), as may mRNA molecules and complementary DNA molecules (e.g. cDNA molecules).
  • Manipulation of the DNA encoding a polypeptide is a particularly powerful technique for both modifying a polypeptide and for generating large quantities of a polypeptide for purification purposes. This may involve the use of PCR techniques to amplify a desired nucleic acid sequence.
  • sequence data provided herein can be used to design primers for use in PCR so that a desired sequence can be targeted and then amplified to a high degree.
  • primers will be at least five nucleotides long and will generally be at least ten nucleotides long (e.g. fifteen to twenty-five nucleotides long). In some cases, primers of at least thirty or at least thirty-five nucleotides in length may be used.
  • hybridising nucleic acid molecules of the present invention can be used as anti-sense molecules to alter the expression of MKK3 polypeptides by binding to complementary nucleic acid molecules. This technique can be used in anti-sense therapy.
  • a hybridising nucleic acid molecule of the present invention may have a high degree of sequence identity along its length with a nucleic acid molecule within the scope of (i)-(v) above (e.g. at least 50%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 98% sequence identity).
  • a nucleic acid molecule within the scope of (i)-(v) above (e.g. at least 50%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 98% sequence identity).
  • the higher the sequence identity a given single stranded nucleic acid molecule has with another nucleic acid molecule the greater the likelihood that it will hybridise to a nucleic acid molecule which is complementary to that other nucleic acid molecule under appropriate conditions.
  • MKK3 nucleic acid molecules may be used as agents for the prophylaxis and/or treatment of inflammatory diseases e.g. asthma, such as in anti
  • nucleic acid molecules for use in the present invention may have one or more of the following characteristics: 1) they may be DNA or RNA;
  • they may be provided in substantially pure form. Thus they may be provided in a form which is substantially free from contaminating polypeptides and/or from other nucleic acids;
  • Nucleic acids comprising a sequence encoding a MKK3 polypeptide or functional variant thereof may be administered by way of gene therapy (see for example Hoshida T. et al, 2002, Pancreas, 25:111-121; fl ⁇ mo Y., 2002, Invest. Ophthalmol. Vis. Sci., 200243:2406-2411; Bollard C, 2002, Blood, 99:3179-3187; Lee E., 2001, Mol. Med., 7:773-782).
  • Gene therapy refers to administration to a subject of an expressed or expressible nucleic acid. Any of the methods for gene therapy available in the art can be used according to the present invention.
  • Delivery of the therapeutic nucleic acid into a patient can be direct in vivo gene therapy (i.e. the patient is directly exposed to the nucleic acid or nucleic acid-containing vector) or indirect ex vivo gene therapy (i.e. cells are first transformed with the nucleic acid in vitro and then transplanted into the patient).
  • an expression vector containing the nucleic acid is administered in such a manner that it becomes intracellular; i.e. by infection using a defective or attenuated retroviral or other viral vectors as described, for example in U.S. 4,980,286 or by Robbins et al, 1998, Pharmacol. Ther., 80:35-47.
  • retroviral vectors that are known in the art are such as those described in Miller et al, (1993, Meth. Enzymol., 217:581-599) which have been modified to delete those retroviral sequences which are not required for packaging of the viral genome and subsequent integration into host cell DNA.
  • adenoviral vectors can be used which are advantageous due to their ability to infect non-dividing cells and such high-capacity adenoviral vectors are described in Kochanek, 1999, Human Gene Therapy, 10:2451-2459.
  • Chimeric viral vectors that can be used are those described by Reynolds et al, 1999, Molecular Medicine Today, 1:25-31.
  • Hybrid vectors can also be used and are described by Jacoby et al, 1997, Gene Therapy, 4: 1282-1283.
  • Direct injection of naked DNA or through the use of microparticle bombardment (e.g. Gene Gun®; Biolistic, Dupont) or by coating it with lipids can also be used in gene therapy.
  • Cell-surface receptors/transfecting compounds or through encapsulation in liposomes, microparticles or microcapsules or by administering the nucleic acid in linkage to a peptide which is known to enter the nucleus or by administering it in linkage to a ligand predisposed to receptor-mediated endocytosis (Wu & Wu, 1987, J. Biol. Chem., 262:4429-4432) can be used to target cell types which specifically express the receptors of interest.
  • nucleic acid ligand compound comprising a nucleic acid
  • the ligand comprises a fusogenic viral peptide designed so as to disrupt endosomes, thus allowing the nucleic acid to avoid subsequent lysosomal degradation.
  • the nucleic acid can be targeted in vivo for cell specific endocytosis and expression by targeting a specific receptor such as that described in WO92/06180, W093/14188 and WO93/20221.
  • the nucleic acid can be introduced intracellularly and incorporated within the host cell genome for expression by homologous recombination (see Zijlstra et al, 1989, Nature, 342:435-428).
  • a gene is transferred into cells in vitro using tissue culture and the cells are delivered to the patient by various methods such as injecting subcutaneously, application of the cells into a skin graft and the intravenous injection of recombinant blood cells such as haematopoietic stem or progenitor cells.
  • Cells into which a nucleic acid can be introduced for the purposes of gene therapy include, for example, epithelial cells, endothelial cells, keratinocytes, fibroblasts, muscle cells, hepatocytes and blood cells.
  • the blood cells that can be used include, for example, T-lymphocytes, B-lymphocytes, monocytes, macrophages, neutrophils, eosinophils, megakaryotcytes, granulocytes, haematopoietic cells or progenitor cells, and the like.
  • Another aspect of the present invention provides a method of screening for and/or diagnosis of an inflammatory disease, e.g. asthma, in a subject and/or monitoring the effectiveness of anti- inflammatory, e.g. asthma, therapy in a subject, which method comprises the step of detecting and/or quantifying in a biological sample obtained from said subject, a MKK3 polypeptide or a MKK3 nucleic acid.
  • the method of detection may comprise detcting MKK3 polypeptide or MKK3 nucleic acid per se, an increase in MKK3 relative to control being indicative of an inflammatory disease.
  • the method of detection may comprise quantification of at least one isoform of a MKK3 polypeptide.
  • the method may comprise detection of the MKK3 isoform 1 and/or the MKK3 isoform 2.
  • a "biological sample” can be obtained from any source, such as a blood or serum sample, or a tissue sample, e.g. leucocytes or lymphocytes.
  • the subject may be a mammal and is preferably a human.
  • a MKK3 polypeptide may be detected using 2D gel electrophoresis.
  • a further convenient means for detecting/quantifying a MKK3 polypeptide involves the use of antibodies.
  • the modified MKK3 polypeptide is detected and/or quantified using an antibody that binds to one or more MKK3 polypeptides.
  • binding of antibody in a biological sample can be used to detect aberrant polypeptide localisation or an aberrant level of increased polypeptide.
  • an "aberrant level” means a level that is increased compared with the level in a subject free from an inflammatory disease e.g. asthma or a reference level.
  • an antibody to a polypeptide as defined herein can be used to assay a patient sample (e.g. a lung biopsy, BAL sample or eosinophil preparation) for the level of the polypeptide where an aberrant level of polypeptide is indicative of an inflammatory disease e.g. asthma.
  • Suitable immunoassays include, without limitation, competitive and non-competitive assay systems using techniques such as western blots, radioimmunoassays, ELISA (enzyme linked immunosorbent assay), "sandwich” immunoassays, immunoprecipitation assays, precipitin reactions, gel diffusion precipitin reactions, immunodiffusion assays, agglutination assays, complement-fixation assays, immunoradiometric assays, fluorescent immunoassays and protein A immunoassays.
  • competitive and non-competitive assay systems using techniques such as western blots, radioimmunoassays, ELISA (enzyme linked immunosorbent assay), "sandwich” immunoassays, immunoprecipitation assays, precipitin reactions, gel diffusion precipitin reactions, immunodiffusion assays, agglutination assays, complement-fixation assays, immunoradiometric assays, fluorescent immunoassays and
  • kits comprising a capture reagent (e.g. an antibody) against a MKK3 polypeptide as defined above.
  • a capture reagent e.g. an antibody
  • a kit may optionally comprise one or more of the following: 1) instructions for using the capture reagent for diagnosis, prognosis, therapeutic monitoring or any combination of these applications;
  • the anti-polypeptide capture reagent itself can be labelled with a detectable marker, e.g. a chemiluminescent, enzymatic, fluorescent, or radioactive moiety.
  • a detectable marker e.g. a chemiluminescent, enzymatic, fluorescent, or radioactive moiety.
  • a MKK3 nucleic acid molecule can also be used in hybridisation assays.
  • a MKK3 nucleic acid molecule comprising at least 8 nucleotides can be used as a hybridisation probe.
  • Hybridisation assays can be used for detection, prognosis, diagnosis, or monitoring of inflammatory diseases e.g. asthma. Accordingly, such a hybridisation assay comprises: i) contacting a patient sample containing nucleic acid with a nucleic acid probe capable of hybridising to aMKK3 nucleic acid, under conditions such that hybridisation can occur; and ii) detecting or measuring any resulting hybridisation.
  • hybridising molecules are at least 10 nucleotides in length and are preferably at least 25 or at least 50 nucleotides in length. More preferably, the hybridising nucleic acid molecules specifically hybridise to nucleic acids within the scope of (i) to (v) above. Most preferably, the hybridisation occurs under stringent hybridisation conditions.
  • stringent hybridisation conditions is where attempted hybridisation is carried out at a temperature of from about 35°C to about 65°C using a salt solution of about 0.9M.
  • stringent hybridisation conditions is where attempted hybridisation is carried out at a temperature of from about 35°C to about 65°C using a salt solution of about 0.9M.
  • the skilled person will be able to vary such conditions as appropriate in order to take into account variables such as probe length, base composition, type of ions present, etc.
  • relatively stringent conditions such as low salt or high temperature conditions
  • highly stringent conditions include hybridisation to filter-bound DNA in 0.5M NaHP0 4 , 7% sodium dodecyl sulphate (SDS), lmM EDTA at 65°C, and washing in O.lxSSC/0.1% SDS at 68°C (Ausubel F.M. et al, eds., 1989, Current Protocols in Molecular Biology, Vol. I, Green Publishing Associates, Inc., and John Wiley & Sons, Inc., New York, at p. 2.10.3).
  • SDS sodium dodecyl sulphate
  • O.lxSSC/0.1% SDS at 68°C
  • Moderately stringent conditions include washing in 0.2xSSC/0.1% SDS at 42°C (Ausubel et al, 1989, supra).
  • Hybridisation conditions can also be rendered more stringent by the addition of increasing amounts of formamide, to destabilise the hybrid duplex.
  • particular hybridisation conditions can be readily manipulated, and will generally be chosen as appropriate.
  • convenient hybridisation temperatures in the presence of 50% formamide are: 42°C for a probe which is 95-100% identical to the fragment of a gene encoding a polypeptide as defined herein, 37°C for 90-95% identity and 32°C for 70-90% identity.
  • the invention also provides a diagnostic kit comprising a nucleic acid probe capable of hybridising to RNA encoding a MKK3 polypeptide, suitable reagents and instructions for use.
  • a diagnostic kit comprising in one or more containers a pair of primers (e.g. each in the size range of 6-30 nucleotides, more preferably 10-30 nucleotides and still more preferably 10-20 nucleotides) that under appropriate reaction conditions can prime amplification of at least a portion of a MKK3 nucleic acid molecule, such as by polymerase chain reaction (se e.g. Innis et al, 1990, PCR Protocols, Academic Press, Inc., San Diego, CA), ligase chain reaction (see EP 320,308) use of Q ⁇ replicase, cyclic probe reaction, or other methods known in the art.
  • primers e.g. each in the size range of 6-30 nucleotides, more preferably 10-30 nucleotides and still more preferably 10-20 nucleotides
  • primers are at least five nucleotides long and will generally be at least ten nucleotides long (e.g. fifteen to twenty-five nucleotides long). In some cases, primers of at least thirty or at least thirty-five nucleotides in length may be used.
  • a MKK3 polypeptide can be used in raising antibodies, e.g. for use in the diagnostic methods described above.
  • the present invention provides an antibody, functionally-active fragment, derivative or analogue thereof, that specifically binds to one or more MKK3 polypeptides.
  • a MKK3 polypeptide, its variants thereof may be used as an immunogen to generate antibodies which immunospecifically bind such an immunogen.
  • Antibodies include, but are not limited to polyclonal, monoclonal, bispecific, humanised or chimeric antibodies, single chain antibodies, Fab fragments and F(ab') fragments, fragments produced by a Fab expression library, anti- idiotypic (anti-Id) antibodies, and epitope-binding fragments of any of the above.
  • the immunoglobulin molecules of the invention can be of any class (e.g. IgG, IgE, IgM, IgD and IgA) or subclass of immunoglobulin molecule.
  • screening for the desired antibody can be accomplished by techniques known in the art, e.g. ELISA (enzyme-linked immunosorbent assay).
  • ELISA enzyme-linked immunosorbent assay
  • to select antibodies, which recognise a specific domain of a polypeptide one may assay generated hybridomas for a product that binds to a polypeptide fragment containing such domain.
  • For selection of an antibody that specifically binds a first polypeptide homologue but which does not specifically bind to (or binds less avidly to) a second polypeptide homologue one can select on the basis of positive binding to the first polypeptide homologue and a lack of binding to (or reduced binding to) the second polypeptide homologue.
  • any technique which provides for the production of antibody molecules by continuous cell lines in culture may be used.
  • the hybridoma technique originally developed by Kohler & Milstein, 1975, Nature, 256:495-497), as well as the trioma technique, the human B-cell hybridoma technique (Kozbor et al, 1983, Immunology Today, 4:72), and the EBN-hybridoma technique to produce human monoclonal antibodies Colde et al, 1985, in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., 77-96).
  • Such antibodies may be of any immunoglobulin class including IgG, IgM, IgE, IgA, IgD and any subclass thereof.
  • the hybridoma producing the mAbs may be cultivated in vitro or in vivo.
  • monoclonal antibodies can be produced in germ- free animals utilising known technology.
  • the mAbs include but are not limited to human mAbs and chimeric mAbs (e.g. human-mouse chimeras).
  • a "chimeric antibody” is a molecule in which different portions are derived from different animal species, such as those having a human immunoglobulin constant region and a variable region derived from a murine mAb. (See, e.g. U.S. 4,816,567; and U.S. 4,816,397).
  • “Humanised antibodies” are antibody molecules from non-human species having one or more complementarity determining regions (CDRs) from the non-human species and a framework region from a human immunoglobulin molecule. (See, e.g. U.S. 5,585,089).
  • Chimeric and humanised mAbs can be produced by recombinant D ⁇ A techniques known in the art, for example using methods described in WO 87/02671; EP 184,187; EP 171,496; EP 173,494; WO 86/01533; U.S. 4,816,567; EP 125,023; Better et al, 1988, Science, 240: 1041-1043; Liu et al,
  • transgenic mice which are incapable of expressing endogenous immunoglobulin heavy and light chain genes, but which can express human heavy and light chain genes.
  • the transgenic mice are immunised in the normal fashion with a selected antigen, e.g. all or a portion of a polypeptide for use in the invention.
  • mAbs directed against the antigen can be obtained using conventional hybridoma technology.
  • the human immunoglobulin transgenes harboured by the transgenic mice rearrange during B cell differentiation, and subsequently undergo class switching and somatic mutation.
  • Completely human antibodies which recognise a selected epitope, can be generated using a technique referred to as "guided selection.”
  • a selected non-human monoclonal antibody e.g. a mouse antibody, is used to guide the selection of a completely human antibody recognising the same epitope.
  • Antibodies can also be generated using various phage display methods known in the art.
  • phage display methods functional antibody domains are displayed on the surface of phage particles which carry the polynucleotide sequences encoding them.
  • phage can be utilised to display antigen-binding domains expressed from a repertoire or combinatorial antibody library (e.g. human or murine).
  • Phage expressing an antigen binding domain that binds the antigen of interest can be selected or identified with antigen, e.g. using labelled antigen or antigen bound or captured to a solid surface or bead.
  • Phage used in these methods are typically filamentous phage including fd and M13 binding domains expressed from phage with Fab, Fv or disulphide stabilised Fv antibody domains recombinantly fused to either the phage gene III or gene NIII polypeptide.
  • Phage display methods that can be used to make the antibodies for use in the present invention include those disclosed in Brinkman et al, J. Immunol. Methods, 182: 41-50, 1995; Ames et al, J. Immunol. Methods, 184:177-186, 1995; Kettleborough et al, Eur. J.
  • the antibody coding regions from the phage can be isolated and used to generate whole antibodies, including human antibodies, or any other desired antigen binding fragment, and expressed in any desired host, including mammalian cells, insect cells, plant cells, yeast, and bacteria, e.g. as described in detail below.
  • Fab, Fab' and F(ab')2 fragments can also be employed using methods known in the art such as those disclosed in WO 92/22324; Mullinax et al., BioTechniques, 12(6): 864-869, 1992; and Sawai et al, AJRI, 34:26-34, 1995; and Better et al, Science, 240: 1041-1043, 1988.
  • Bispecific antibodies can be made by methods known in the art. Traditional production of full length bispecific antibodies is based on the coexpression of two immunoglobulin heavy chain-light chain pairs, where the two chains have different specificities (Milstein et al, 1983, Nature, 305:537-539). Because of the random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of 10 different antibody molecules, of which only one has the correct bispecific structure. Purification of the correct molecule, which is usually done by affinity chromatography steps, is rather cumbersome, and the product yields are low. Similar procedures are disclosed in WO 93/08829, and in Traunecker et al, 1991, EMBO J., 10:3655-3659.
  • antibody variable domains with the desired binding specificities are fused to immunoglobulin constant domain sequences.
  • the fusion preferably is with an immunoglobulin heavy chain constant domain, comprising at least part of the hinge, CH2, and CH3 regions. It is preferred to have the first heavy-chain constant region (CHI) containing the site necessary for light chain binding, present in at least one of the fusions.
  • DNAs encoding the immunoglobulin heavy chain fusions and, if desired, the immunoglobulin light chain are inserted into separate expression vectors, and are co-transfected into a suitable host organism.
  • the bispecific antibodies may be composed of a hybrid immunoglobulin heavy chain with a first binding specificity in one arm and a hybrid immunoglobulin heavy chain-light chain pair (providing a second binding specificity) in the other arm. It was found that this asymmetric structure facilitates the separation of the desired bispecific agent from unwanted immunoglobulin chain combinations, as the presence of an immunoglobulin light chain in only one half of the bispecific molecule provides for a facile way of separation. This approach is disclosed in WO 94/04690. For further details for generating bispecific antibodies see, for example, Suresh et al, Methods in Enzymology, 1986, 121:210.
  • the invention may utilise fimctionally-active fragments, derivatives or analogues of the anti- polypeptide immunoglobulin molecules.
  • “Functionally-active” means that the fragment, derivative or analogue is able to elicit anti-anti-idiotype antibodies (i.e. tertiary antibodies) that recognise the same antigen that is recognised by the antibody from which the fragment, derivative or analogue is derived.
  • the antigenicity of the idiotype of the immunoglobulin molecule may be enhanced by deletion of framework and CDR sequences that are C-terminal to the CDR sequence that specifically recognises the antigen.
  • synthetic peptides containing the CDR sequences can be used in binding assays with the antigen by any binding assay method known in the art.
  • the invention provides antibody fragments such as, but not limited to, F(ab')2 fragments and Fab fragments.
  • Antibody fragments which recognise specific epitopes may be generated by known techniques.
  • F(ab')2 fragments consist of the variable region, the light chain constant region and the CHI domain of the heavy chain and are generated by pepsin digestion of the antibody molecule.
  • Fab fragments are generated by reducing the disulphide bridges of the F(ab')2 fragments.
  • the invention also provides heavy chain and light chain dimers of the antibodies for use in the invention, or any minimal fragment thereof such as Fvs or single chain antibodies (SCAs) (e.g. as described in U.S.
  • Single chain antibodies are formed by linking the heavy and light chain fragments of the Fv region via an amino acid bridge, resulting in a single chain polypeptide. Techniques for the assembly of functional Fv fragments in E. coli may be used (Skerra et al, 1988, Science, 242:1038-1041).
  • the invention provides fusion proteins of the immunoglobulins of the invention (or functionally active fragments thereof), for example in which the immunoglobulin is fused via a covalent bond (e.g. a peptide bond), at either the N-terminus or the C-terminus to an amino acid sequence of another polypeptide (or portion thereof, preferably at least 10, 20 or 50 amino acid portion of the polypeptide) that is not the immunoglobulin.
  • a covalent bond e.g. a peptide bond
  • the immunoglobulin, or fragment thereof is covalently linked to the other polypeptide at the N-terminus of the constant domain.
  • such fusion proteins may facilitate purification, increase half-life in vivo, and enhance the delivery of an antigen across an epithelial barrier to the immune system.
  • the immunoglobulins of the invention include analogues and derivatives that are either modified, i.e. by the covalent attachment of any type of molecule as long as such covalent attachment that does not impair immunospecific binding.
  • the derivatives and analogues of the immunoglobulins include those that have been further modified, e.g. by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatisation by known protecting/blocking groups, proteolytic cleavage, linkage to a cellular ligand or other polypeptide, etc. Any of numerous chemical modifications may be carried out by known techniques, including, but not limited to specific chemical cleavage, acetylation, formylation, etc. Additionally, the analogue or derivative may contain one or more non-classical amino acids.
  • the foregoing antibodies can be used in methods known in the art relating to the localisation and activity of the MKK3 polypeptides, e.g. for imaging or radioimaging these polypeptides, measuring levels thereof in appropriate physiological samples, in diagnostic methods, etc. and for radiotherapy.
  • the antibodies can be produced by any method known in the art for the synthesis of antibodies, in particular, by chemical synthesis or by recombinant expression, and are preferably produced by recombinant expression technique.
  • a nucleic acid encoding the antibody may be assembled from chemically synthesised oligonucleotides (e.g. as described in Kutmeier et al, 1994, BioTechniques, 17:242), which, briefly, involves the synthesis of overlapping oligonucleotides containing portions of the sequence encoding antibody, annealing and ligation of those oligonucleotides, and then amplification of the ligated oligonucleotides by PCR.
  • the nucleic acid encoding the antibody may be obtained by cloning the antibody. If a clone containing the nucleic acid encoding the particular antibody is not available, but the sequence of the antibody molecule is known, a nucleic acid encoding the antibody may be obtained from a suitable source (e.g. an antibody cDNA library, or cDNA library generated from any tissue or cells expressing the antibody) by PCR amplification using synthetic primers hybridisable to the 3' and 5' ends of the sequence or by cloning using an oligonucleotide probe specific for the particular gene sequence.
  • a suitable source e.g. an antibody cDNA library, or cDNA library generated from any tissue or cells expressing the antibody
  • antibodies specific for a particular antigen may be generated by any method known in the art, for example, by immunising an animal, such as a rabbit, to generate polyclonal antibodies or, more preferably, by generating monoclonal antibodies.
  • a clone encoding at least the Fab portion of the antibody may be obtained by screening Fab expression libraries (e.g. as described in Huse et al, 1989, Science, 246: 1275-1281) for clones of Fab fragments that bind the specific antigen or by screening antibody libraries (See, e.g. Clackson et al, 1991, Nature, 352:624; Hane et al, 1997, Proc. Natl. Acad. Sci. USA, 94:4937).
  • nucleic acid encoding at least the variable domain of the antibody molecule may be introduced into a vector containing the nucleotide sequence encoding the constant region of the antibody molecule (see e.g. WO 86/05807; WO 89/01036; and U.S. 5,122,464).
  • Vectors containing the complete light or heavy chain for co-expression with the nucleic acid to allow the expression of a complete antibody molecule are also available.
  • the nucleic acid encoding the antibody can be used to introduce the nucleotide substitution(s) or deletion(s) necessary to substitute (or delete) the one or more variable region cysteine residues participating in an intrachain disulphide bond with an amino acid residue that does not contain a sulphydryl group.
  • Such modifications can be carried out by any method known in the art for the introduction of specific mutations or deletions in a nucleotide sequence, for example, but not limited to, chemical mutagenesis, PCR based methods, in vitro site directed mutagenesis (Hutchinson et al., 1978, J. Biol. Chem., 253:6551), etc.
  • techniques developed for the production of "chimeric antibodies" (Morrison et al,
  • a chimeric antibody is a molecule in which different portions are derived from different animal species, such as those having a variable region derived from a murine mAb and a human antibody constant region, e.g. humanised antibodies.
  • the vector for the production of the antibody molecule may be produced by recombinant DNA technology using techniques well known in the art. Methods, which are well known to those skilled in the art, can be used to construct expression vectors containing an antibody molecule coding sequences and appropriate transcriptional and translational control signals. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination.
  • the expression vector is transferred to a host cell by conventional techniques and the transfected cells are then cultured by conventional techniques to produce an antibody.
  • the host cells used to express a recombinant antibody may be either bacterial cells such as Escherichia coli, or, preferably, eukaryotic cells, especially for the expression of whole recombinant antibody molecule.
  • mammalian cells such as Chinese hamster ovary cells (CHO), in conjunction with a vector such as the major intermediate early gene promoter element from human cytomegalovirus is an effective expression system for antibodies (Foecking et al, 1988, Gene8 45:101; Cockett et al, 1990, BioTechnology ⁇ 8:2).
  • host-expression vector systems may be utilised to express an antibody.
  • Such host- expression systems represent vehicles by which the coding sequences of interest may be produced and subsequently purified, but also represent cells which may, when transformed or transfected with the appropriate nucleotide coding sequences, express the antibody molecule in situ.
  • These include but are not limited to microorganisms such as bacteria (e.g. E. coli, B. subt ⁇ lis) transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing antibody coding sequences; yeast (e.g.
  • Saccharomyces, Pichia transformed with recombinant yeast expression vectors containing antibody coding sequences; insect cell systems infected with recombinant virus expression vectors (e.g. baculovirus) containing the antibody coding sequences; plant cell systems infected with recombinant virus expression vectors (e.g. cauliflower mosaic virus, CaMN; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g. Ti plasmid) containing antibody coding sequences; or mammalian cell systems (e.g.
  • COS COS, CHO, BHK, HEK 293, 3T3 cells harbouring recombinant expression constructs containing promoters derived from the genome of mammalian cells (e.g. metallothionein promoter) or from mammalian viruses (e.g. the adenovirus late promoter; the vaccinia virus 7.5K promoter).
  • mammalian cells e.g. metallothionein promoter
  • mammalian viruses e.g. the adenovirus late promoter; the vaccinia virus 7.5K promoter.
  • a number of expression vectors may be advantageously selected depending upon the use intended for the antibody molecule being expressed. For example, when a large quantity of such a polypeptide is to be produced, vectors which direct the expression of high levels of fusion protein products that are readily purified may be desirable.
  • vectors include, but are not limited, to the E. coli expression vector pUR278 (Ruther et al, 1983, EMBO J.
  • pGEX vectors may also be used to express foreign polypeptides as fusion proteins with glutathione S- transferase (GST).
  • fusion proteins are soluble and can easily be purified from lysed cells by adsorption and binding to a matrix glutathione-agarose beads followed by elution in the presence of free glutathione.
  • the pGEX vectors are designed to include thrombin or factor Xa protease cleavage sites so that the cloned target gene product can be released from the GST moiety.
  • Autographa californica nuclear polyhedrosis virus (Ac ⁇ PN) is used as a vector to express foreign genes.
  • the virus grows in Spodopterafrugiperda cells.
  • the antibody coding sequence may be cloned individually into non-essential regions (e.g. the polyhedrin gene) of the virus and placed under control of an Ac ⁇ PV promoter (e.g. the polyhedrin promoter).
  • an Ac ⁇ PV promoter e.g. the polyhedrin promoter
  • a number of viral-based expression systems e.g. an adenovirus expression system
  • a host cell strain may be chosen which modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired. Such modifications (e.g. glycosylation) and processing (e.g. cleavage) of polypeptide products may be important for the activity of the polypeptide.
  • cells lines that stably express an antibody can be produced by transfecting the cells with an expression vector comprising the nucleotide sequence of the antibody and the nucleotide sequence of a selectable (e.g. neomycin or hygromycin), and selecting for expression of the selectable marker.
  • a selectable e.g. neomycin or hygromycin
  • Such engineered cell lines may be particularly useful in screening and evaluation of agents s that interact directly or indirectly with the antibody molecule.
  • the expression levels of the antibody molecule can be increased by vector amplification (for a review, see Bebbington and Hentschel, The use of vectors based on gene amplification for the expression of cloned genes in mammalian cells in D ⁇ A cloning, Vol.3. (Academic Press, New York, 1987)).
  • vector amplification for a review, see Bebbington and Hentschel, The use of vectors based on gene amplification for the expression of cloned genes in mammalian cells in D ⁇ A cloning, Vol.3. (Academic Press, New York, 1987)).
  • a marker in the vector system expressing antibody is amplifiable
  • increase in the level of inhibitor present in culture of host cell will increase the number of copies of the marker gene. Since the amplified region is associated with the antibody gene, production of the antibody will also increase (Crouse et al, 1983, Mol. Cell. Biol., 3:257).
  • the host cell may be co-transfected with two expression vectors the first vector encoding a heavy chain derived polypeptide and the second vector encoding a light chain derived polypeptide.
  • the two vectors may contain identical selectable markers which enable equal expression of heavy and light chain polypeptides.
  • a single vector may be used which encodes both heavy and light chain polypeptides. In such situations, the light chain should be placed before the heavy chain to avoid an excess of toxic free heavy chain (Proudfoot, 1986, Nature, 322:52; Kohler, 1980, Proc. Natl. Acad. Sci. USA, 77:2197).
  • the coding sequences for the heavy and light chains may comprise cDNA or genomic DNA.
  • the antibody molecule may be purified by any method known in the art for purification of an antibody molecule, for example, by chromatography (e.g. ion exchange chromatography, affinity chromatography such as with protein A or specific antigen, and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for the purification of polypeptides.
  • chromatography e.g. ion exchange chromatography, affinity chromatography such as with protein A or specific antigen, and sizing column chromatography
  • centrifugation e.g. ion exchange chromatography, affinity chromatography such as with protein A or specific antigen, and sizing column chromatography
  • differential solubility e.g., differential solubility, or by any other standard technique for the purification of polypeptides.
  • any fusion protein may be readily purified by utilising an antibody specific for the fusion protein being expressed.
  • a system described by Janknecht et al. allows for the ready purification of non-denatured fusion proteins expressed in human cell lines (Janknecht et al, 1991, Proc. Natl. Acad. Sci. USA, 88:8972-897).
  • the gene of interest is subcloned into a vaccinia recombination plasmid such that the open reading frame of the gene is translationally fused to an amino-terminal tag consisting of six histidine residues.
  • the tag serves as a matrix binding domain for the fusion protein.
  • Extracts from cells infected with recombinant vaccinia virus are loaded onto Ni 2+ nitriloacetic acid-agarose columns and histidine-tagged polypeptides are selectively eluted with imidazole-containing buffers.
  • antibodies or fragments thereof are conjugated to a diagnostic moiety.
  • the antibodies can be used for diagnosis or to determine the efficacy of a given treatment regimen. Detection can be facilitated by coupling the antibody to a detectable substance.
  • detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, radioactive nuclides, positron emitting metals (for use in positron emission tomography), and non-radioactive paramagnetic metal ions. See generally U.S. 4,741,900 for metal ions which can be conjugated to antibodies for use as diagnostics according to the present invention.
  • Suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase; suitable prosthetic groups include streptavidin, avidin and biotin; suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride and phycoerythrin; suitable luminescent materials include luminol; suitable bioluminescent materials include luciferase, luciferin, and aequorin; and suitable radioactive nuclides include 125 1, 131 I, ⁇ n In, and 99 Tc.
  • Antibodies or fragments thereof can also be conjugated to a therapeutic agent or drug moiety to modify a given biological response.
  • the therapeutic agent or drug moiety is not to be construed as limited to classical chemical therapeutic agents.
  • the drug moiety may be a polypeptide possessing a desired biological activity.
  • Such polypeptides may include, for example, a toxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin; a polypeptide such as tumour necrosis factor, -interferon, ⁇ -interferon, nerve growth factor, platelet derived growth factor, tissue plasminogen activator, a thrombotic agent or an anti-angiogenic agent, e.g.
  • angiostatin or endostatin or, a biological response modifier such as a lymphokine, interleukin-1 (IL-1), interleukin-2 (IL-2), interleukin-6 (IL-6), granulocyte macrophage colony stimulating factor (GM-CSF), granulocyte colony stimulating factor (G-CSF), nerve growth factor (NGF) or other growth factor.
  • IL-1 interleukin-1
  • IL-2 interleukin-2
  • IL-6 interleukin-6
  • GM-CSF granulocyte macrophage colony stimulating factor
  • G-CSF granulocyte colony stimulating factor
  • NGF nerve growth factor
  • an antibody can be conjugated to a second antibody to form an antibody heteroconjugate as described in U.S. 4,676,980.
  • An antibody with or without a therapeutic moiety conjugated to it can be used as a therapeutic that is administered alone or in combination with cytotoxic factor(s) and/or cytokine(s).
  • kits comprising an antibody against a MKK3 polypeptide.
  • a kit may optionally comprise one or more of the following: (1) instructions for using the antibody for diagnosis, prognosis, therapeutic monitoring or any combination of these applications; (2) a labelled binding partner to the antibody; (3) a solid phase (such as a reagent strip) upon which the antibody is immobilised; and (4) a label or insert indicating regulatory approval for diagnostic, prognostic or therapeutic use or any combination thereof.
  • the anti-polypeptide antibody itself can be labelled with a detectable marker, e.g. a chemiluminescent, enzymatic, fluorescent, or radioactive moiety.
  • Figure 1 shows the amino acid sequence of MKK3 variants A, B and C. The tandem spectra used to identify MKK3 from lymphocytes is shown in bold.
  • Figure 2 shows the nucleic acid sequence of MKK3 variant A.
  • Figure 3 shows an image obtained from 2-dimensional electrophoresis of a sample prepared from lymphocytes, which has been annotated to identify twelve landmark features, designated CD4TL1 to CD4TL11 and CD4TL13, and which are illustrative of an embodiment of an aspect of the present invention.
  • Figure 4 shows the differential expression of MKK3 seen between asthma samples and control samples in lymphocytes.
  • Figures 5+6 show the quantitative analysis of MKK3 mRNA in human tissues.
  • MKK3 was isolated by 2D-electrophoresis from lymphocytes Sample preparation
  • Lymphocyte preparation Blood was drawn from donors by antecubital venepuncture into lithium heparin and mixed with 1/3 volume sterile 6% dextran solution. Erythrocytes were allowed to sediment under gravity for 45 min at room temperature, after which leukocyte-rich supernatants were layered onto 10 ml cushions of Ficoll-Paque (1.077 g/ml) and centrifuged at 800 g for 25 min at room temperature. The granulocyte pellet was washed twice in phosphate buffered saline and re-suspended at a density of 10 9 /ml in PBS containing 5 mM EDTA, 0.1% NaN 3 and 1% BSA (PBS-BSA).
  • the cell suspension was mixed at a ratio of 3:2 with CD8 MACS microbeads (MACS, Miltenyi Biotec) and incubated at 4°C, with intermittent mixing, for 30 min, after which the volume was made up to 1 ml with PBS-BSA.
  • the cell/bead mixture was loaded onto a steel fiber matrix column placed in a strong magnetic field and the column was washed at a low flow rate with 10 volumes of ice-cold PBS-BSA to elute CD8- cells (predominantly lymphocytes).
  • the CD8-preparation was washed twice with PBS and residual erythrocytes removed by hypotonic lysis.
  • Cells are re-suspended in RPMI-BSA and cell counts and viability are determined in trypan blue-treated samples using a haemocytometer. Purity was assessed in cytocentrifuge preparations stained with May-Gr ⁇ nwald Giemsa stain.
  • Isoelectric focusing was performed using the Immobiline7 DryStrip Kit (Pharmacia BioTech), following the procedure described in the manufacturer's instructions, see Instructions for Immobiline7 DryStrip Kit, Pharmacia, # 18-1038-63, Edition AB.
  • Immobilised pH Gradient (IPG) strips (18cm, pH 3-10 non-linear strips; Pharmacia Cat. # 17-1235-01) were rehydrated overnight at 20°C in a solution of 8M urea, 2% (w/v) CHAPS, lOmM DTT, 2% (v/v) Resolytes 3.5-10, as described in the Immobiline DryStrip Users Manual.
  • the current limit was set to 10mA for 12 gels, and the wattage limit to 5W.
  • the temperature was held at 20°C throughout the run.
  • the strips were immediately removed and immersed for lOmin at 20°C in a first solution of the following composition: 6M urea; 2% (w/v) DTT; 2% (w/v) SDS; 30% (v/v) glycerol (Fluka 49767); 0.05M Tris/HCl, pH 6.8 (Sigma Cat T-1503).
  • the strips were removed from the first solution and immersed for lOmin at 20°C in a second solution of the following composition: 6M urea; 2% (w/v) iodoacetamide (Sigma 1-6125); 2% (w/v) SDS; 30% (v/v) glycerol; 0.05M Tris/HCl, pH 6.8.
  • the strips were loaded onto supported gels for SDS-PAGE according to Hochstrasser et al, 1988, Anal. Biochem. 173:412-423, with modifications as specified below.
  • the gels were cast between two glass plates of the following dimensions: 23cm wide x 24cm long (back plate); 23cm wide x 24cm long with a 2cm deep notch in the central 19cm (front plate).
  • the back plate was treated with a 0.4% solution of ⁇ - methacryl-oxypropyltrimethoxysilane in ethanol (BindSilaneTM; Pharmacia Cat. No. 17-1330-01).
  • the front plate was treated with a 2% solution of dimethyldichlorosilane dissolved in octamethyl cyclo-octasilane (RepelSilaneTM Pharmacia Cat. No. 17-1332-01) to reduce adhesion of the gel.
  • the dried plates were assembled into a casting box with a capacity of thirteen gel sandwiches.
  • the front and back plates of each sandwich were spaced by means of 1mm thick spacers, 2.5cm wide.
  • the sandwiches were interleaved with acetate sheets to facilitate separation of the sandwiches after gel polymerization.
  • Casting was then carried out according to Hochstrasser et al, op. cit.
  • a 9-16% linear polyacrylamide gradient was cast (second dimension gel), extending up to a point 2cm below the level of the notch in the front plate, using the Angelique gradient casting system (Large Scale Biology).
  • Stock solutions were as follows. Acrylamide (40% in water) was from Serva (Cat. # 10677).
  • the cross-linking agent was PDA (BioRad 161-0202), at a concentration of 2.6% (w/w) of the total starting monomer content.
  • the gel buffer was 0.375M Tris/HCl, pH 8.8.
  • the polymerisation catalyst was 0.05% (v/v) TEMED (BioRad 161-0801), and the initiator was 0.1%
  • a solution of 0.5% (w/v) agarose (Fluka Cat 05075) was prepared in running buffer (0.025M Tris, 0.198M glycine (Fluka 50050), 1% (w/v) SDS, supplemented by a trace of bromophenol blue).
  • the agarose suspension was heated to 70°C with stirring, until the agarose had dissolved.
  • the top of the supported second dimension gel was filled with the agarose solution, and the equilibrated strip was placed into the agarose, and tapped gently with a palette knife until the gel was intimately in contact with the second dimension gel.
  • the gels were placed in the second dimension running tank, as described by Amess et al, 1995, Electrophoresis 16: 1255-1267.
  • the tank was filled with running buffer (as above) until the level of the buffer was just higher than the top of the region of the second dimension gel s which contained polyacrylamide, so as to achieve efficient cooling of the active gel area.
  • Running buffer was added to the top buffer compartments formed by the gels, and then voltage was applied immediately to the gels using a Consort E-833 power supply.
  • the gels were run at 20mA/gel.
  • the wattage limit was set to 150W for a tank containing 6 gels, and the voltage limit was set to 600V.
  • the gels were then run at 40mA gel, with the same voltage and wattage limits as before, until the bromophenol blue line was 0.5cm from the bottom of the gel.
  • the temperature of the buffer was held at 16°C throughout the run. Gels were not run in duplicate.
  • a computer-readable output was produced by imaging the fluorescently stained gels with a scanner as described in WO 01/63294.
  • This scanner has a gel carrier with four integral fluorescent markers (designated Ml, M2, M3, M4) that are used to correct the image geometry and are a quality control feature to confirm that the scanning has been performed correctly.
  • the gels were removed from the stain, rinsed with water and allowed to air dry briefly, and imaged on the scanner. After imaging, the gels were sealed in polyethylene bags containing a small volume of staining solution, and then stored at 4°C.
  • the output from the scanner was first processed using the MELAN1E7 II 2D PAGE analysis program (Release 2.2, 1997, BioRad Laboratories, Hercules, California, Cat. No. 170-7566) to autodetect the registration points, Ml, M2, M3 and M4; to autocrop the images (i.e. to eliminate signals originating from areas of the scanned image lying outside the boundaries of the gel, e.g. the reference frame); to filter out artefacts due to dust; to detect and quantify features; and to create image files in GIF format.
  • Features were detected using the following parameters:
  • Landmark identification was used to determine the pi and MW of features detected in the images. Twelve landmark features, designated CD4TL1 to CD4TL11 and CD4TL13, were identified in a sample image of lymphocytes. These landmark features are identified in Figure 3 and were assigned the pi and/or MW values identified in Table 1.
  • each feature in the study gels was then assigned a pi value by linear interpolation or extrapolation (using the MELANIE7-II software) to the two nearest landmarks, and was assigned a MW value by linear interpolation or extrapolation (using the MELANIE7-II software) to the two nearest landmarks.
  • the fundamental principle in multi-resolution modelling is that smooth signals may be modelled as an evolution through "scale space", in which details at successively finer scales are added to a low resolution approximation to obtain the high resolution signal.
  • This type of model is applied to the flow field of vectors (defined at each pixel position on the reference image) and allows flows of arbitrary smoothness to be modelled with relatively few degrees of freedom.
  • Each image is first reduced to a stack, or pyramid, of images derived from the initial image, but smoothed and reduced in resolution by a factor of 2 in each direction at every level (Gaussian pyramid) and a corresponding difference image is also computed at each level, representing the difference between the smoothed image and its progenitor (Laplacian pyramid).
  • the Laplacian images represent the details in the image at different scales.
  • the warping process brought about good alignment between the common features in the primary master image, and the images for the other samples.
  • the MELANIE7 II 2D PAGE analysis program was used to calculate and record approximately 500-700 matched feature pairs between the primary master and each of the other images.
  • the accuracy of this program was significantly enhanced by the alignment of the images in the manner described above.
  • all pairings were finally examined by eye in the MelView interactive editing program and residual recognisably incorrect pairings were removed. Where the number of such recognisably incorrect pairings exceeded the overall reproducibility of the method (as measured by repeat analysis of the same biological sample) the gel selected to be the primary master gel was judged to be insufficiently representative of the study gels to serve as a primary master gel. In that case, the gel chosen as the primary master gel was rejected, and different gel was selected as the primary master gel, and the process was repeated.
  • a composite master image was thus generated by initialising the primary master image with its feature descriptors. As each image was transformed into the primary master geometry, it was digitally summed pixel by pixel into the composite master image, and the features that had not been paired by the procedure outlined above were likewise added to the composite master image description, with their centroids adjusted to the master geometry using the flow field correction.
  • MCI molecular cluster index
  • this MCI profile was traceable to the actual stored gel from which it was generated, so that proteins identified by computer analysis of gel profile databases could be retrieved.
  • the LEVIS also permitted the profile to be traced back to an original sample or patient.
  • the Wilcoxon Rank-Sum test was test was performed between the control and the asthma samples for each MCI.
  • the MCIs which recorded a p-value less than or equal to 0.05 were selected as statistically significant with 95% selectivity.
  • Polypeptides were robotically excised from the 2D gel and processed to generate tryptic digest peptides. Tryptic peptides were analysed by mass spectrometry using a PerSeptive Biosystems Voyager-DETM STR Matrix-Assisted Laser Desorption lonization Time-of-Flight (MALDI-TOF) mass spectrometer, and selected tryptic peptides were analysed by tandem mass spectrometry (MS/MS) using a Micromass Quadrupole Time-of-Flight (Q-TOF) mass spectrometer (Micromass, Altrincham, U.K.) equipped with a nanoflowTM electrospray Z-spray source.
  • MALDI-TOF PerSeptive Biosystems Voyager-DETM STR Matrix-Assisted Laser Desorption lonization Time-of-Flight
  • MS/MS tandem mass spectrometry
  • Q-TOF Micromass Quadrupole Time-of-Flight
  • amino acid sequences of tryptic digest peptides of MKK3 were determined from matches to tryptic digest peptides of P46734, see Figure 1.
  • sequences were identified using peptide mass data derived from mass spectrometer analysis and the MOWSE database search procedure.
  • Peptide mass information can provide a 'fingerprint' signature sufficiently discriminating to allow for the unique and rapid identification of unknown sample proteins, independent of other analytical methods such as protein sequence analysis. Practical experience has shown that sample proteins can be uniquely identified using as few as 3- 4 experimentally determined peptide masses when screened against a fragment database derived from over 50,000 proteins (D.J.C. Pappin, P. Hojrup and AJ.
  • the version of the code used for a MOWSE database search had the following modifications: the size of the parent protein is not included in the calculation such that large proteins such as titin no longer bias the score; instead the theoretical frequency of a peptide of mass (x) is estimated using the mass (x) of the peptide and the mean mass of an amino acid whilst allowing for a probability of 0.2 for a missed internal cleavage (by trypsin) and 0.1 for the probability of the occurrence of a proteolytic cleavage site.
  • the score assigned to a match on a peptide mass (x) is the logarithm of the probability of finding such a match at random and is inversely proportional to the frequency of fragments of that mass.
  • the score is thus calculated using the linear regression formula found by plotting the score for a match on peptide mass (x) against the mass (x).
  • Mass matches to predicted trypsin fragments were identified in this manner for MKK3, which also matched to P46734, see Figure 1. The accuracy of this method was 20 ppm.
  • Example 2 MKK3 is differentially expressed in asthma samples
  • MKK3 had an apparent MW of 38,658 Da, and an apparent pi of 7.31 (see Table 2, the predicted MW and pi values for this protein are 36,171 Da and 6.20 respectively). In asthma samples a 3.65 fold increase in the expression of MKK3 was seen relative to the control samples ( Figure 4).
  • MKK3 or upstream regulators thereof are putative targets for therapeutic intervention of inflammatory diseases such as respiratory diseases, e.g. asthma.
  • Example 3 Expression of MKK3 mRNA Quantitative analysis of MKK3 mRNA was performed using methods well known to those skilled in the art. The expression of the three splice variants of MKK3 in 26 human tissues was investigated. Variant A showed high expression in brain and kidney whereas Variant B is expressed ubiquitously. Variant C showed profound expression in trachea. Further detailed analysis indicated that Variant C is upregulated in lung primary fibroblast cells and epithelial cells upon IL-4 or TNF- ⁇ stimulation, suggesting its specific roles in the inflammatory pathology of the respiratory system ( Figures 5 and 6).
  • MKK3 is involved in the development of asthma pathology.
  • MKK3 or upstream regulators thereof are putative targets for therapeutic intervention in imflammatory diseases such as respiratory diseases, e.g. asthma.
  • the present invention is not to be limited in terms of the particular embodiments described in this application, which are intended as single illustrations of individual aspects of the invention. Functionally equivalent methods and apparatus within the scope of the invention, in addition to those enumerated herein, will be apparent to those skilled in the art from the foregoing description and accompanying drawings. Such modifications and variations are intended to fall within the scope of the appended claims.

Abstract

L'invention concerne des méthodes de traitement de maladies inflammatoires, notamment de maladies respiratoires, telles que l'asthme et des états pathologiques associés à l'asthme. Ces méthodes consistent à réguler la kinase 3 de la protéine kinase activée par des agents mitogènes (MKK3) d'un polypeptide, ces agents modulant l'expression ou l'activité du polypeptide. L'invention concerne des méthodes d'identification de ces agents et d'utilisation de MKK3 dans le diagnostic de maladies inflammatoires, notamment de maladies respiratoires telles que l'asthme et des états pathologiques associés à l'asthme.
PCT/GB2003/004477 2002-10-15 2003-10-15 Regulation de mkk3 WO2004035139A1 (fr)

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Publication number Priority date Publication date Assignee Title
CN114258317A (zh) * 2019-06-18 2022-03-29 斯克里普斯研究学院 用于治疗炎性病症的方法和组合物

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